The Structural Properties,digestion Characteristics And Immunoregulatory Activity Of Ovotransferrin And Lactoferrin

As a high-abundance protein in egg white(accounting for about 12% of the total protein content),ovotransferrin(OVT)belongs to the transferrin family with human serum transferrin and lactoferrin(HLF),bovine lactoferrin(BLF)as well as melanin transferrin,and has high sequence homology and similar three-dimensional structure with BLF and HLF.A large number of studies have shown that OVT,BLF and HLF have similar biological activities,such as antibacterial,anti-virus,anti-tumor,immunomodulatory,iron absorption promoting activities.Due to the low output of lactoferrin extraction and purification in China,the weak key technology of industrialization,and the long-term dependence on imports of lactoferrin products,it is an urgent direction to explore and work hard to find products that can replace lactoferrin while breaking through the technical barrier of large-scale lactoferrin extraction and purification.Based on the similarity in structure and functional activities,OVT has the potential to be used as a substitute for lactoferrin.However,no comprehensive and indepth comparative analyses has been conducted on the structural characteristics,digestive characteristics,allergenicity and functional activity of OVT,BLF and HLF,and the feasibility of substitution remains to be confirmed.Therefore,by analyzing the primary and advanced structures,glycosylation status and sites of the three transferrins,we can seek the potential relationship between their structural and digestive characteristics,allergenicity and immunoregulatory activity,identify the sensitization risks of the three transferrins and their digestive products,and explore the cellular immunoregulatory activity and related molecular mechanisms of the three transferrins and their digestive products,which can be used as the feasibility basis for the application of OVT and its enzymatic products as a substitute for lactoferrin in infant formula or special medical foods,and help to solve the needs of the transferrins industry.The main research contents and results of this topic are as follows:(1)Physicochemical properties,homology,secondary structure,glycosylation and phosphorylation modification sites,three-dimensional structure,protein interaction network and functional annotation of tri-source transferrin were analyzed by bioinformatics prediction.It was found that OVT had 52% sequence homology and 70%3D structure similarity with BLF and HLF.The isoelectric points of the three transferrins were 6.69,8.67 and 8.47,respectively,with high similarity in secondary structure and lobular domain.They all have a certain number of glycosylation sites and a high degree of phosphorylation,and interact with nearly 10 important proteins in the body,suggesting that they are also involved in some important biological processes.(2)The high-level structure of three-source transferrin was analyzed by a variety of spectroscopic methods,and the three sources were evaluated by X-ray diffraction,turbidity,fluorescence spectroscopy,circular dichroism and nanoscale DSC.Thermal stability of source transferrin at different p H and ionic strength.At the level of secondary structure,the arrangement of OVT and BLF is similar,while HLF has higherα-helix content and lower β-sheet content.On the tertiary structure,due to the different microenvironments of the tyrosine,tryptophan and phenylalanine residues of the three proteins in their respective three-dimensional structures,the presented UV spectra and endogenous fluorescence spectra exist visible difference.In the solution system,OVT has a larger particle size and PDI,showing a significantly different particle size distribution,while BLF and HLF are more hydrophobic than OVT.The denaturation temperatures of OVT,BLF,and HLF were 80.64°C,65.71°C,and 90.01°C,respectively,and the denaturation temperatures of OVT and BLF decreased significantly under acidic conditions.The α-helix and β-turn contents of the three proteins all showed a decreasing trend after heat treatment at different p H,while the β-sheet contents increased significantly.The endogenous fluorescence properties and turbidity of the three protein solutions were also affected by p H and heating conditions.(3)Based on UHPLC-ESI-MS/MS and protein glycosylation database,combined with bioinformatics,the N/O glycosylation sites and sugar chain types of ternary transferrin were identified and analyzed.Two N-glycosylation sites were found in OVT,BLF and HLF,N492/N637,N252/N495 and N156/N497,respectively.In terms of Nglycan chain composition,OVT and BLF contain 40 and 9 sugar chains,respectively,mostly short sugar chains,while HLF contains 112 sugar chains,mostly long sugar chains.They all contain four kinds of sugar chains of hexose,N-acetylhexosaminose,fucose and sialic acid.Compared with OVT and BLF,HLF is more abundant in Nglycan species,and has a higher proportion of fucose and sialic acid residues in the sugar chain composition.The results of O-glycosylation analysis showed that OVT had15 O-glycosylation sites and contained 123 O-glycosyl chains,of which the short chains were significantly more than the long chains,and showed higher fucose and sialylation modifications.There are only two O-glycosylation sites and four O-glycosyl chains in BLF,all of which exist in the form of short sugar chains.A total of 9 O-glycosylation sites were identified by HLF,including 14 O-glycosyl chains,mainly short ones.Among them,OVT was significantly more than BLF and HLF in the number of glycosylation sites and types of sugar chains.(4)Using Tricine-SDS-PAGE,dynamic light scattering,Nano-HPLC-MS/MS,amino acid analysis and high-power microscopy,combined with the results of glycosylation analysis,the gastrointestinal tract of adults and infants with tri-source transferrin was analyzed.Simulated digestion products were analyzed to obtain information such as peptide sequence and molecular weight,free amino acid content and type,particle size distribution,and microscopic morphology.The electrophoresis spectra showed that the band distributions of the adult and infant digestive products of the three proteins were significantly different.High-magnification microscopy and particle size distribution characterization showed that the particle shapes and sizes of the adult and infant digestive products were different.Among adult digestive products,OVT and BLF digestive products had a greater variety of free amino acids,but BLF digests had significantly higher levels of several typical amino acids.Among the digestive products of infants and young children,BLF has the most types of free amino acids and higher content of various amino acids,while the types and contents of OVT are the least.The composition of the digested products was analyzed by Nano LCMS/MS,and it was found that the number of peptides identified by OVT was the largest in the adult digested products,with 62 peptides,and 35 and 36 peptides were identified by BLF and HLF,respectively.OVT,BLF and HLF identified 41,42 and 62 peptides,respectively,in the digestive products of infants.The peptide information of the digested product was compared with the identified peptides containing glycosylation sites,and it was found that OVT,BLF and HLF adult digestive products contained 26,2 and 3 glycopeptides,respectively,and their infant digestive products contained 3,4and 3 glycopeptides,respectively.(5)Based on the RBL-2H3 cell sensitization model mediated by IgE,the cell degranulation degree,β-hexosaminidase release rate,histamine and trypsin release amount were determined after induction of cells by different samples,and combined with three transfections.The antigenic epitope sequence of ferritin was used to evaluate and analyze the allergenicity of tri-source transferrin and its digestion products.The results showed that,from the perspective of the overall change level,the indexes of the infant digested products showed the highest level after induction of cells,the index levels of the adult digested products group were in the middle,and the three intact proteins showed the lowest cell degranulation,β-hexosaminidase release rates,histamine and trypsin release rates.Combining the three protein digestion properties and epitope data analysis,the occultation of epitopes in the intact protein structure and the low degree of digestibility of the infant digestate led to their differences in allergenicity.Interms of the sensitization level,I-OVT≈I-BLF(27)I-HLF,A-OVT≈ABLF(27)A-HLF,BLF(27)OVT(27)HLF.(6)The expression of MHC Ⅱ,CD80 and CD86 molecules on the surface of dendritic cells as well as cytokines IL-12 and TNF-α level induced by different samples were measured by immunofluorescence and ELISA,respectively.It was found that the expression of three costimulatory molecules was up-regulated and the levels of two cytokines were significantly increased in the treatment group.Among them,the infant digestive products from the three transferrin have the strongest promoting effect on the expression of costimulatory molecules and the secretion of cytokines,which may be related to the type and number of peptide fragments contained in infant digestive products with binding capacity on MHC class II molecule,while the intact proteins and their adult digestive products have relatively weak promoting effects.Further,induced dendritic cells were co-cultured with T lymphocytes in contact mode at a co-culture ratio of 1:4,1:9 and 1:19.It was found that induced dendritic cells could promote the proliferation of T lymphocyte and the secretion of related cytokines(IFN-γ、 IL-4),and thus promote T lymphocytes to differentiate into Th1 cells.Finally,by constructing the co-culture system of induced dendritic cells,T lymphocytes and macrophages,it was found that Th1 cells promoted by induced dendritic cells and their cytokines would activate macrophages to secrete NO and IL-6,thus mediating the cellular immune process.In general,the infant digestive products from the three transferrins have the strongest cellular immunomodulatory activity,followed by adult digestive products,and the weakest intact proteins.Interms of immunomodulatory activity,I-BLF(27)IHLF(27)I-OVT,A-BLF(27)A-OVT≈A-HLF,OVT(27)BLF≈HLF.Overall,the comparative analysis of the primary/advanced structures,glycosylation structures,digestion properties,allergenicity and cellular immune regulatory activity of the three transferrins showed that OVT is feasible to replace BLF and HLF,but further research is still needed to promote the implementation of alternative plans.